Thyroid hormone (TH) plays critical roles in the differentiation, growth, and metabolism of virtually all tissues and its dysregulation causes some of the most prevalent endocrine diseases. Thyroid hormone receptors (TRs) are ligand-regulatable transcription factors that bind to thyroid hormone response elements (TREs) in the promoters of target genes and regulate their transcription. Unliganded TRs bind to corepressors and repress basal transcription by recruiting histone deacetylase (HDACs). Liganded TRs recruit co-activator complexes that acetylated neighboring histones and interact with general transcriptional factors. TH also can negatively-regulate some target genes but the molecular mechanisms are not well understood. Our preliminary studies showed that co-factors are differentially recruited to target genes suggesting target genes may have unique "signature" co-activator profiles. However, the molecular details of how TR isoforms, co-repressors, and co-activators are recruited to individual target genes have not been characterized significantly. We thus plan to investigate the temporal recruitment pattern of endogenous TR isoforms and co-factors on TREs of positively- and negatively-regulated target genes using chromatin immunoprecipitation (ChIP) assays while assessing their functional significance by performing simultaneous nuclear run-on studies. The Specific Aims of this proposal are: 1) To study the recruitment of endogenous co-repressors, HDACs, and co-activators as well as in vitro transcriptional activity of several positively-regulated target genes in a Ts-responsive pituitary cell line that contains endogenous TRalpha and TRbeta isoforms; 2) To determine the critical co-factors for TH-regulated transcription on the target genes by using siRNAs as well as histone acetylation and deacetylation inhibitors in conjunction with ChIP and nuclear run-on assays; and 3) To study the molecular mechanism of Ts-mediated negative-transcription by employing permanently transfected and transformed pituitary cell lines expressing thyroid stimulating hormone beta (TSHbeta) or alpha-subunit glycoprotein hormone (alpha-sub) as well as identify novel co-factors involved in ligand-mediated transcription.